Liver Plasma Membranes Research Articles

Ultrastructural liver changes in the experimental thyrotoxicosis.

Aim of the study is to evaluate ultrastructural changes of rat liver in experimental thyrotoxicosis. For the study, 36 male rats have been utilized, weighing approximately 150-190 g, which were divided into three groups: the first, control group (12 animals) was composed of healthy rats that received intragastric sodium chloride 0.9% solution, the second group (12 animals) - animals with experimental thyrotoxicosis, which received intragastric solution of L-thyroxine at the rate of 200 μg/kg for 2 weeks, and the third group (12 animals) - rats with experimental thyrotoxicosis, which received intragastric solution of L-thyroxine at the rate of 200 μg/kg for 4 weeks. For electron-microscopic studies small pieces of liver tissue were taken at the end of the 2nd and 4th weeks of the experiment. The material was studied and documented in electron micrographs by using a TEM-125K electron microscope. In experiment in white male rats the electron-microscopic state of the liver in thyrotoxicosis has been studied. It has been established that thyrotoxicosis is accompanied by the significant changes of the hepatocytes ultrastructure, blood and bile capillaries. Experimental thyrotoxicosis causes significant damage of the liver plasma membranes and intracellular structural components of hepatocytes and endothelial cells. In experimental thyrotoxicosis, on the background of microcirculatory disorders, significant damage of plasmatic and intracellular organoid membranes of hepatocytes in the liver develops, which has an adverse effect on the functionality of the organ. The found ultrastructural changes are aggravated depending on the duration of thyrotoxicosis.

Anticardiolipin antibody in egyptian patients with chronic hepatitis c in correlation with liver injury

Background The presence of anticardiolipin (ACL) antibodies in chronic hepatitis C may be owing to induction of neoantigens by chronic viral infection, which may be because of disruption of liver cell membranes. The occurrence of these ACL antibodies may be observed because of recognition of these neoantigens by the immune system. Aim The aim of the work was to study the prevalence and clinical significance of ACL antibodies in chronic hepatitis C infection and their relationship with disease progression. Participants and methods Our study was performed on 90 individuals recruited from Gastroenterology and Hepatology Outpatient Clinics and Internal Medicine Department of Kafr Sheikh Institute. The participants were divided into the following three groups: group A consisted of 30 patients with chronic hepatitis C virus (HCV) infection with compensated liver cirrhosis, group B consisted of 30 patients with chronic HCV infection with decompensated liver cirrhosis, and group C consists of 30 individuals as control. All patients and control were subjected to full history taking, full clinical examination, complete blood count, liver function tests, renal profile, pelvis–abdominal ultrasound, and serum levels of ACL immunoglobulin G and α-fetoprotein. Results The results showed that seven (23%) of the HCV-positive patients in group A and 18 (60%) of the HCV-positive patients in group B were ACL immunoglobulin G-positive in comparison with none in the control group (0%). Conclusion The prevalence of ACL antibodies is increased during HCV infection, and HCV should be regarded as a possible causative factor in the antiphospholipid syndrome.

Pim-1L Protects Cell Surface-Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level.

ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [3H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoprotein-targeted therapy.

Chronic effects of hydro-alcoholic artemisia absinthium extract on the liver enzymes and tissue changes of adult mal rat

Artemisia absinthium has many pharmacological effects, but toxic effects of it, were seen on nervous system and liver. Therefore, the aim of this study was to evaluate the chronic effects of different doses of Artemisia absinthium extract on the enzymes and histopathological changes of the liver tissue of adult normal male rat. Method and materials: In this experimental study, forty eight male Wistar rats were randomly divided into 6 groups of 8 as follows: Control, sham (recipient of distilled water) and 4 experimental groups that received Artemisia absinthium hydro- alcoholic extract at doses of 125, 250, 500 and 1000 mg/kg intraperitoneally. The data were analyzed using one-way ANOVA and Duncan post hoc tests. P≤0.05 was considered statistically significant. Results: After 8 weeks, doses of 125, 250 and 500 mg/kg could significantly reduce amount of ALT, AST and ALP. Dose of 1000 mg/kg increased ALT, AST and ALP. From the standpoint of histopathological study, doses of 125, 250 and 500 Artemisia absinthium had no significant side effect on liver tissue, but 1000 mg/kg caused destruction of liver cell membranes, enlargement of sinusoidal space, sporadic leukocyte infiltration, Kupffer cells hypertrophy, and vascular congestion. Conclusion: Maximum dose of Artemisia absinthium extract (1000 mg/kg) increased liver enzymes and destroy liver tissues of normal male rats. Keywords: Artemisia Absinthium, Enzymes, Histopathology, Liver, Rat

Study on functional damage mechanism of the hepatocellular mitochondria in the rats with sepsis

Objective To study the effect on the expression of the aquaporin 8 (AQP8) in inner mitochondrial membrane of the liver cells in the rats with sepsis, and on the function of the liver cells and mitochondria. Methods 24 SD rats were divided into the control group and the septic group with 12 rats respectively. The sepsis model of the rats was established by the cecal ligation and puncture. After 18 h of moulding, killing the rats and taking the blood, the alanime aminotransferase (ALT), aspartate aminotransferase (AST) and mitochondrial aspartate aminotransferase (m-AST) were detected. The content of Adenosine Triphosphate (ATP) in the liver cells, mitochondrial membrane potential levels, and the activity of the mitochondrial membrane Na+ -K+ -ATPase, Mg2+ -ATPase, Ca2+ -ATPase and Ca2+ -Mg2+ -ATPase were tested. The expression quantity of AQP8 and AQP8 mRNA were measured with Western blotting and Real-time quantitative polymerase chain reaction (Real-time PCR). Results Compared with the control group, the serum ALT, AST and m-AST was higher that in the septic group and the content of ATP in the liver cells was lower significantly [(233.02±110.16) U/L vs. (29.81±13.16) U/L, (742.56±441.41) U/L vs. (99.78±41.67) U/L, (412.78±252.56) U/L vs. (46.23±24.24) U/L, (19.27±8.76) nmol/mg vs. (94.65±17.79) nmol/mg, P<0.01), the activity of the ATPases of the mitochondria and the mitochondrial membrane potential level was decreased significantly [(2.81±0.81) U/mgprot vs. (4.74±0.84) U/mgprot, (2.59±1.03) U/mgprot vs. (4.49±0.73) U/mgprot, (1.63±1.26) U/mgprot vs. (3.48±0.43) U/mgprot, (2.54±0.97) U/mgprot vs. (4.68±0.81) U/mgprot, 1.73±1.06 vs 7.65±3.54, P<0.01], the expression quantity of AQP8 and the transcription quantity of AQP8 mRNA were reduced (0.68±0.03 vs. 1.24±0.05, 0.34±0.19 vs. 1.00, P<0.01). Conclusion The reduction of the expression quantity of AQP8 in hepatocellular mitochondrial membrane and dysfunction of the ionic channel in sepsis, result in the significant reduction of the mitochondrial membrane potential level and content of ATP in the liver cell, and acute damage to the liver cells finally. Key words: Sepsis; Liver; Mitochondrial; Aquaporin 8

Intrahepatic and extrahepatic aminotransferase elevation associated with clinical‐therapeutic events in a schizophrenic patient

Key Clinical MessageA schizophrenic patient showed rhabdomyolysis with idiopathic transaminitis. The intermixed pattern of intrahepatic and extrahepatic alanine aminotransferase (ALT) elevation is associated with respective clinical‐therapeutic events. Aminotransferases play a role as surrogate biomarkers of “liver metabolic functioning” beyond the obsolete classical concept associating ALT elevation only with liver cellular damage.

Mechanisms of iron hepatotoxicity

Mechanisms of iron hepatotoxicity

Update on a tumor-associated NADH oxidase in gastric cancer cell growth.

Gastric cancer is one of the most common human malignancies, and its prevalence has been shown to be well-correlated with cancer-related deaths worldwide. Regrettably, the poor prognosis of this disease is mainly due to its late diagnosis at advanced stages after the cancer has already metastasized. Recent research has emphasized the identification of cancer biomarkers in the hope of diagnosing cancer early and designing targeted therapies to reverse cancer progression. One member of a family of growth-related nicotinamide adenine dinucleotide (NADH or hydroquinone) oxidases is tumor-associated NADH oxidase (tNOX; ENOX2). Unlike its counterpart CNOX (ENOX1), identified in normal rat liver plasma membranes and shown to be stimulated by growth factors and hormones, tNOX activity purified from rat hepatoma cells is constitutively active. Its activity is detectable in the sera of cancer patients but not in those of healthy volunteers, suggesting its clinical relevance. Interestingly, tNOX expression was shown to be present in an array of cancer cell lines. More importantly, inhibition of tNOX was well correlated with reduced cancer cell growth and induction of apoptosis. RNA interference targeting tNOX expression in cancer cells effectively restored non-cancerous phenotypes, further supporting the vital role of tNOX in cancer cells. Here, we review the regulatory role of tNOX in gastric cancer cell growth.

Impaired Hepatic Uptake by Organic Anion-Transporting Polypeptides Is Associated with Hyperbilirubinemia and Hypercholanemia in Atp11c Mutant Mice.

Biliary excretion of organic anions, such as bile acids (BAs), is the main osmotic driving force for bile formation, and its impairment induces intrahepatic cholestasis. We investigated the involvement of Atp11c in the hepatic transport of organic anions using Atp11c mutant mice, which exhibit hypercholanemia and hyperbilirubinemia. Pharmacokinetic analysis following a constant intravenous infusion in Atp11c mutant mice showed decreased hepatic sinusoidal uptake and intact biliary secretion of [(3)H]17β estradiol 17β-d-glucuronide. Consistent with this result, compared with cells and membranes from control mice, isolated hepatocytes, and liver plasma membranes from Atp11c mutant mice had a much lower uptake of [(3)H]17β estradiol 17β-d-glucuronide and expression of organic anion-transporting polypeptides, which are transporters responsible for hepatic uptake of unconjugated BAs and organic anions, including bilirubin glucuronides. Uptake of [(3)H]TC into hepatocytes and expression of Na(+)-taurocholate cotransporting polypeptide in liver plasma membranes, which mediates hepatic uptake of conjugated BAs, was also lower in the Atp11c mutant mice. Bile flow rate, biliary BA concentration, and expression of hepatobiliary transporters did not differ between Atp11c mutant mice and control mice. These results suggest that Atp11c mediates the transport of BAs and organic anions across the sinusoidal membrane, but not the canalicular membrane, by regulating the abundance of transporters. Atp11c is a candidate gene for genetically undiagnosed cases of hypercholanemia and hyperbilirubinemia, but not of intrahepatic cholestasis. This gene may influence the pharmacological and adverse effect of drugs because organic anion-transporting polypeptides regulate their systemic exposure.

Cell communication across gap junctions: a historical perspective and current developments.

Collaborative communication lies at the centre of multicellular life. Gap junctions (GJs) are surface membrane structures that allow direct communication between cells. They were discovered in the 1960s following the convergence of the detection of low-resistance electrical interactions between cells and anatomical studies of intercellular contact points. GJs purified from liver plasma membranes contained a 27kDa protein constituent; it was later named Cx32 (connexin 32) after its full sequence was determined by recombinant technology. Identification of Cx43 in heart and later by a further GJ protein, Cx26 followed. Cxs have a tetraspan organization in the membrane and oligomerize during intracellular transit to the plasma membrane; these were shown to be hexameric hemichannels (connexons) that could interact end-to-end to generate GJs at areas of cell-to-cell contact. The structure of the GJ was confirmed and refined by a combination of biochemical and structural approaches. Progress continues towards obtaining higher atomic 3D resolution of the GJ channel. Today, there are 20 and 21 highly conserved members of the Cx family in the human and mouse genomes respectively. Model organisms such as Xenopus oocytes and zebra fish are increasingly used to relate structure to function. Proteins that form similar large pore membrane channels in cells called pannexins have also been identified in chordates. Innexins form GJs in prechordates; these two other proteins, although functionally similar, are very different in amino acid sequence to the Cxs. A time line tracing the historical progression of wide ranging research in GJ biology over 60years is mapped out. The molecular basis of channel dysfunctions in disease is becoming evident and progress towards addressing Cx channel-dependent pathologies, especially in ischaemia and tissue repair, continues.

The studies on the toxicity mechanism of environmentally hazardous natural (IAA) and synthetic (NAA) auxin – The experiments on model Arabidopsis thaliana and rat liver plasma membranes

This paper concerns the studies towards membrane-damage effect of two auxins: indole-3-acetic acid – IAA and 1-naphthaleneacetic acid – NAA on plant (Arabidopsis thaliana) and animal (rat liver) model membranes. The foregoing auxins are plant growth regulators widely used in agriculture to control the quality of the crop. However, their accumulation in the environment makes them hazardous for the living organisms. The aim of our investigations was to compare the effect of natural (IAA) vs. synthetic (NAA) auxin on the organization of plant and animal model membranes and find a possible correlation between membrane-disturbing effect of these compounds and their toxicity. The collected data evidenced that auxins cause destabilization of membranes, decrease their condensation and weakens interactions of molecules. The alterations in the morphology of model systems were also noticed. The foregoing effects of auxins are concentration-dependent and additionally NAA was found to act on animal vs. plant membranes more selectively than IAA. Interestingly, both IAA and NAA induce the strongest disordering in model lipid system at the concentration, which is frequently reported as toxic to animal and plants. Based on the above findings it was proposed that membrane-damage effect induced by IAA and NAA may be important from the point of view of the mechanism of toxicity of these compounds and cannot be ignored in further investigations in this area.

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system.

English

ABSTRACT Pharmacodynamical studies showed that most drugs elicit their effects by acting on 3 kinds of protein molecules known as receptors, enzymes or transporters. Although their detail properties had not been explained for decades the roles of transporters in drug kinetics and dynamics has been well understood, even have been applied in the therapy. Transporters are classified into 2 major classes, the solute carriers (SLC) and ATP-binding cassette (ABC) families. SLC transporters do not possess ATP binding site property as those of ABC transporters. SLC transporters consist of 3 SLC subfamilies i.e. organic cation transporters (OCTs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs). In contrast, ABC transporters require ATP hydrolysis to transport substrate across cell membrane. Human ABC-transporters consist of ABCA1- 13, ABCB1-11, ABCC1-12, ABCD1-4, ABCE1, ABCF1-3 and ABCG1-8 subfamily. Although the originally funtion of transporter is to transport specific physiological substrate such as nutrient, hormone, cytokines, neurotransmitters and other physiological subtances across cell membrane the specificity is not restricted to each substrate. Drugs and other xenobiotics which have structural similarity to the physiological substrates are recognized and transported by the related transporters. The competition of them on transporters therefore may lead to the occurence of drug-drug interactions (DDI) or drugphysiological substrate interaction in the drug-kinetics phase. Many transporters located in the liver, intestinal and renal epithelial cell membranes involve in the transport of endogenous substance or xenobiotics including drugs play important roles as protective barrier. Since transporters also serve as the targets of drug action it is understood that transporters play important role in the pathogenesis of diseases as well as in the drug therapy of diseases.

Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles

The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

Membrane adaptation in phospholipids and cholesterol in the widely distributed, freeze-tolerant wood frog, Rana sylvatica

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to -2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.

Protein expression of kidney and liver bilitranslocase in rats exposed to mercuric chloride—A potential tissular biomarker of toxicity

Bilitranslocase (BTL) is a plasma membrane carrier that transports organic anions of physiological and pharmacological interest. It is expressed in basolateral plasma membrane of kidney and liver. BTL has been recently described as a marker of transition from normal tissue to its neoplastic transformation in human kidney. Inorganic mercury is a major environmental contaminant that produces many toxic effects. Previous reports have described an interaction between BTL and mercuric ions. This study was designed to evaluate the renal and hepatic expression of BTL in rats exposed to a nephrotoxic and hepatotoxic dose of HgCl2. Male rats were treated with a single injection of HgCl2 at a dose of 4mg/kg body wt, i.p. (HgCl2 group). Control rats received the vehicle alone (Control group). Studies were carried out 18h after injection. Afterwards, the kidneys and livers were excised and processed for histopathological studies or immunoblot (homogenates and crude membranes) techniques. In rats treated with HgCl2, immunoblotting showed a significant decrease in the abundance of BTL in homogenates and plasma membranes from kidney and liver. BTL decrease of expression might reflect the grade of damage in renal tubule cells and in hepatocytes. Thus, BTL might be postulated as a new biomarker of tissue toxicity induced by mercury.

Intake of Xylooligosaccharides Alters the Structural Organization of Liver Plasma Membrane Bilayer

Xylooligosaccharides (XOS) are non-digestible carbohydrate prebiotics that beneficially affect the host by selective stimulation of specific bacteria in the gastro-intestinal tract. The impact of XOS on gastrointestinal microflora and blood lipids is well known but the exact mechanism of action on liver membranes is still unclear. The organization of membrane lipids in domains is known to be important for the proper functioning of various receptors and mechanisms triggering cell signaling. In this study the influence of XOS-enriched diet on the lipid bilayer structure of rat liver plasma membrane was investigated. XOS intake caused a slight decrease of the fluidity of lipid extracts from liver plasma membranes compared to the controls. This observation was based on the increased generalized polarization (GP) and blue shifted emission spectra of Laurdan. The elevated amount of membrane sphingomyelin may be one possible reason for the reported effects. The micron-scale phase separation of the lipid extracts was also investigated by fluorescence microscopy. A different temperature of phase separation and domain pattern was observed in plasma membrane lipid extracts from XOS-fed animals. We presume that it could be assigned to the altered lipid composition of the membrane bilayer, in particular to the changes in the sphingomyelin/cholesterol ratio. All observed alterations are discussed in the light of the impact of XOS on human health and physiology.

The regulation mechanism of protein kinase Cδ on arsenic liver injury caused by coal-burning

To investigate the effects of mRNA transcriptional and protein expressions of protein kinase Cδ (PKCδ) on the development of arsenic liver injury caused by coal-burning. Population study:133 arsenic exposures were selected as arsenic exposure groups including the ward non-patient group (25 cases) , no obvious hepatopathy group (38 cases) , mild (43 cases) and moderate to severe hepatopathy group (27 cases) from the area with endemic arsenism in Guizhou province. Another 34 healthy residents were selected as the control group in non-arsenic pollution village. The urine and peripheral blood were collected from the subjects. The arsenic contents in urine and mRNA expressions of PKCδ in peripheral blood were detected. Animal experiment study:thirty wistar rats were randomly by random number table divided into control group, drinking water arsenic poisoning group and coal-burning arsenic poisoning group (i.e., low, medium and high arsenic contaminated grain group) by random number table method, including 6 rats in each group. The control group was fed normally for 3 months, drinking water arsenic poisoning group and coal-burning arsenic poisoning groups were fed respectively with 10 mg/kg As2O3 solution and different concentrations (25, 50 and 100 mg/kg) of arsenic-containing feed which was persisted 3 months. The arsenic contents in urine, mRNA expression levels of PKCδ in peripheral blood and liver tissue and the protein expression levels of phosphorylated protein kinase Cδ(pPKCδ) in liver tissue were detected. The median(quartile) of arsenic contents in urine were 25.58 (18.62-40.73), 56.66 (38.93-76.77), 64.90 (39.55- 98.37) and 75.47 (41.30-109.70) µg/g Cr respectively for the non-patient group, no obvious hepatopathy group, mild and moderate to severe hepatopathy group. The levels were higher than that in the control group (23.34 (17.84-37.45) µg/g Cr) (P < 0.05), except for the ward non-patient group. The arsenic contents in rat urine were 2223.61 (472.98-3976.73), 701.16 (194.01-1300.27), 1060.94 (246.33-2585.47) and 3101.11 (1919.97-5407.07) µg/g Cr, respectively for the drinking water arsenic poisoning group, the low, medium and high dosage arsenic grain contamination groups, all higher than that in the control group (94.32 (22.65-195.25) µg/g Cr) (P < 0.05) . The protein expressions of pPKCδ in liver tissue were 324.83 ± 25.06, 278.50 ± 30.57, 308.83 ± 34.67 and 326.33 ± 35.09, which were significantly higher than that in the control group (240.17 ± 28.07) (P < 0.05) . The protein expression levels of pPKCδ in liver cell membrane were 0.49 ± 0.06,0.33 ± 0.05,0.37 ± 0.06 and 0.50 ± 0.08, which were significantly higher than that in the control group (0.28 ± 0.04) (P < 0.05) . The protein expression levels of pPKCδ in liver cell cytoplasm were 0.38 ± 0.06,0.31 ± 0.05, 0.35 ± 0.05 and 0.36 ± 0.05, which were significantly higher than that in the control group (0.24 ± 0.05) (P < 0.05). The arsenic may regulate protein expressions of pPKCδ and induce its membrane translocation, and cause the development of arsenic liver injury caused by coal-burning.

Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F0F1 ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F1 sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F0F1 complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F0F1 complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.

A novel mutation within a transmembrane helix of the bile salt export pump (BSEP,ABCB11) with delayed development of cirrhosis

The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC-2) or progressive familial intrahepatic cholestasis (PFIC-2). The molecular basis of BSEP-associated liver disease in a sibling pair was characterized by immunostaining, gene sequencing, bile salt analysis and recombinant expression in mammalian cells and yeast for localization and in vitro activity studies respectively. Benign recurrent intrahepatic cholestasis was considered in a brother and sister who both suffered from intermittent cholestasis since childhood. Gene sequencing of ABCB11 identified the novel missense mutation p.G374S, which is localized in the putative sixth transmembrane helix of BSEP. Liver fibrosis was present in the brother at the age of 18 with progression to cirrhosis within 3 years. Immunofluorescence of liver tissue showed clear canalicular BSEP expression; however, biliary concentration of bile salts was drastically reduced. In line with these in vivo findings, HEK293 cells showed regular membrane targeting of human BSEP(G374S), whereas in vitro transport measurements revealed a strongly reduced transport activity. The novel mutation p.G374S impairs transport function without disabling membrane localization of BSEP. While all other known BSEP mutations within transmembrane helices are associated with PFIC-2, the new p.G374S mutation causes a transitional phenotype between BRIC-2 and PFIC-2.